Alkaline phosphatase (EC 3.1.3.1, hereinafter also referred to as AP) is an enzyme that catalyzes a reaction for hydrolyzing phosphoric monoester to generate alcohol and inorganic phosphate, and this enzyme is known for its wide distribution both in procaryotes and eucaryotes. In addition to its use as an enzyme for genetic engineering, AP is widely used as a marker enzyme for enzyme immunoassay analysis (EIA). Currently, calf intestinal AP (CIAP) is predominantly used as AP for EIA. One of the reasons for the convenience of CIAP is its high specific activity. Although the specific activity of commercially available CIAP varies depending on the manufacturer or the grade, some high-specific-activity CIAP with a p-nitro phenyl phosphate substrate has a specific activity of more than 6,000 U/mg protein. In addition, various commercially available high-sensitivity luminescent substrates for CIAP containing 1,2-dioxetane or acridan in their basic structure are conducive to high measurement sensitivity in immunoassay analysis.
One of the most important tasks in immunoassay analysis is to ensure high sensitivity. Although several attempts to achieve high sensitivity have been made, including an attempt to increase the number of marker enzyme molecules adsorbed to each molecule of antigen, and an attempt to develop a high-sensitivity substrate of marker enzymes, the present sensitivity of the immunodiagnosis does not fully meet the required sensitivity level. For example, a negative result obtained by immunodiagnosis using an influenza detection kit does not completely eliminate the possibility of infection. Further, considering that the concentration of the target substance in the specimen is often 1 pM or lower, and considering the reduction in time for diagnosis, increasing the sensitivity is a perpetual challenge.
In order to meet the requirement of high sensitivity, skilled artisans have so far attempted a method of isolating a specific type of CIAP having high specific activity from multiple CIAP isozymes during the process of purification, a method of specifying the gene of CIAP having high specific activity and producing a recombinant enzyme of the gene, and a method of increasing specific activity by introducing an amino acid mutation specific to the site critically involved in the increase in specific activity. On the other hand, there have been no reports so far of successful acquisition of AP having specific activity comparable to or greater than calf-derived AP. Further, although the specific activity of AP has been evaluated based on the reactivity to p-nitro phenyl phosphate, i.e., a substrate of standard AP, AP is actually desired to have high reactivity to various luminescent substrates used in actual high-sensitivity immunoassay analysis. However, there have been no successful reports producing AP superior to CIAP in terms of practical usability from a different source.
CIAP also has a problem of poor stability. Although bacterial AP such as E. coli-derived AP cells has higher stability than that of CIAP, its specific activity is significantly poor compared with CIAP. Non-patent Document 1 and Non-patent Document 2 disclose AP derived from the genus Shewanella as an example of AP having relatively high specific activity. However, the specific activities of the disclosed enzymes were all less than 2,000 U/mg. The AP disclosed in Non-patent Document 1 was patented with the approval of its industrial usability (Patent Document 1). However, the enzyme of that invention is characterized by its usefulness for gene engineering technologies because of its poor thermal stability compared with E. coli-derived AP. Thus, the invention nowhere mentions usability of the enzyme as a marker enzyme for immunoassay analysis, or even a possibility of such usage. Further, although Patent Document 2 discloses AP derived from the genus Bacillus, the specific activity of that enzyme is about 3,000 U/mg, which is not considered sufficient. Further, the Bacillus-derived enzyme has low reactivity with respect to 1,2-dioxetane and acridan luminescent substrates. As such, the Bacillus-derived AP is not sufficiently practical.